| Background:Malignant tumors are hazardous to human health,one of the most serious diseases.At the 18th General Assembly on International Union Against Cancer Which is held in 2007,the World Health Organization published a study report shows that the situation of the world's cancer will become increasingly serious,the next 20 years the number of new patients will from the current 10 million increased to 15 million each year,the number of deaths will also be increased from six million to 10 million per year due to cancer.In tumor therapy,chemotherapy is currently one of the important means in clinical tumor treatment,but because most chemotherapy drugs lack of targeting,when destruction of tumor cells at the same time often damaged the body's normal cells.This has led to serious side effects and the low rate of response to chemotherapy.Which has limited its clinical application,so improve drug targeting is one of the effective means to improve the effect of chemotherapy and reducing drug toxicity.The so-called nanometer(nm) or millimic is a length unit,1nm=10-9m(that is, a billionth of a meter),it involves neither the material level in a non-micro-macro also relatively independent of the middle area-referred View(mesoscopy).When the particle size into the nanometer scale,that is,with the quantum size effect,small size effect,surface effect and macroscopic quantum tunneling effect,which showed a number of new substances in different macroscopic properties,such as low melting point,high specific heat capacity,high-expansion coefficient and high reactivity,high proliferation rate,high strength,high toughness,and the peculiar magnetic properties of highly absorbing.Nanotechnology is a new technology,which was recognized as the most promising fields of science of the international 21st century,Research on the use of nano-biotechnology in the medical field has become the hot spot in recent years,at present,nano-technology will be used in the diagnosis and treatment of malignant tumors has been carried out extensive research will be expected to imaging diagnosis,immunology diagnosis,diagnostic biosensors and bio-chip diagnostic areas such as the birth of some new ways in which cancer patients receive more timely and accurate diagnosis.In the treatment,the study found that the use of nano-scale polymer particles as a new drug delivery and controlled release carrier,because of its small size super-micro through targeting the role of positioning and release into the body's cells,of nucleic acids,proteins,etc.material for the molecular level of life treatment,not only improved the efficacy of existing drugs,but also significantly reduced the adverse reactions of their own,the role of nano-drug targeting mechanism in accordance with its three different types:①active targeting,the antibodies or ligand-specific targeting molecules,such as coupling to the surface of particle orientation distribution of drugs to target tissue;②passive targeting,by control and modified the physical or chemical properties of the vector(size,shape, hydro-philicity,surface charge and the wall aperture,etc.) can control its distribution and drug release characteristics in the body,or to choose the nanoparticles which targeted to the body of various organizations and different disease affinity can also achieve the purpose;③physical and chemistry targeting,such as:use of local magnetic field in vitro,with super paramagnetic iron nanoparticles as a carrier at the external magnetic field under the directional distribution in a predetermined target tissue.By changing the physical and chemical properties of the nanoparticles,such as size,mass,charge and water affinity,can reduced non-specific interactions with non-targeting organs,tissues and cells,thus increasing the target position/non-target ratio of the drug.Thus nano controlled-release system in the medical field,especially in the treatment of tumors has been widely studied.For example,liposome parcels adriamycin(trade name:Kai Lai),has been up in the clinical application,but the liposome itself has a certain degree of toxicity,and low encapsulation efficiency,bad stability,therefore,find better carrier material is a research hot spot.Chitosan(CS) is a polycationic polysaccharide derivatives which is widely exist in nature,also known as soluble chitin,chitosan,etc.from D-glucosamine and N-acetyl-D-glucosamine composition,based onβ-(1,4)glycosidic bond linking the straight-chain polysaccharide,the chemical known as(1,4)-2-amino-2-deoxy-β-D-glucose, its structure similar to cellulose,chitosan is non-toxic,has good biocompatibility and degradability,because of its positively charged,make it interaction with negatively charged polymers,macromolecules and even some polyanionic in the liquid medium,which happened the sol-gel transition process making the drug-loaded nanoparticles more easily;From the point of biology pharmacy view,chitosan nanoparticles have the characteristics of attached to the mucosal surface,which makes it especially suited for targeted mucosal drug delivery. Preparation of chitosan controlled-release formulations and lipid micro-spheres absorption which loading chemotherapy drugs,insulin,antibiotics and other drugs, has been achieved good results,some have been applied to clinical,but in practical applications have found that ordinary drug-loaded nanoparticles in the blood vessels easily uptake by reticulo-endothelial system(RES) and rapid clearance and it is difficult to achieve the desired result,if through physical adsorption or covalent binding one layer or multi-layer hydrophilic polymer objects on the surface of the general nano-particles,such as polyethylene glycol(PEG),may form a mushroom-shaped,brush-like shape or steric layer,thereby preventing plasma protein opsonin close chitosan,chitosan reduced plasma protein between the van der Waals and gravitational,so that chitosan nanoparticles can evade the body mononuclear phagocytic system to capture,extend the nanoparticles carrying drugs and their blood circulation time,it is referred to as "stealth" nanoparticles,to overcome the disadvantage of the traditional nanoparticles most rapidly phagocytosis by RES in vivo and difficult to arrive other tumor tissues,thus prolonging circulation time in vivo,through the mechanism of the enhanced permeability and retention effect. In this study,we choice CS as a raw material,using anion cohesion method, prepared chitosan nano-particles which containing the mode drug,and modified by polyethylene glycol,testing its characterization by in vitro cyto-toxicity assay and in vivo anti-tumor experiments,to explore its inhibition of tumor cell proliferation with a view to provide theoretical basis for clinical application.Objective:1.Discussion the feasibility of chitosan nano-particles as a stealth anti-cancer drug delivery system.2.Preparation of the ordinary chitosan nano-particles(CS NPs) and the polyethylene glycol-modified chitosan nano-particles(PEG/CS NPs),and load a model drug epirubicin(EPI),respectively,detection of drug-loaded nanoparticles characterization and in vitro release properties.3.Observation of drug-loaded nanoparticles on tumor cell proliferation inhibitory effect in vitro to prove that drug-loaded chitosan nanoparticles with the same anti-tumor effect compared with free drug.Further verification that the chitosan nanoparticles to be able to play a slow-release effect of the drug.4.Use nude mice for the in vivo anti-tumor tests to prove that the stealth chitosan nanoparticles has better anti-tumor effect in vivo as compared with ordinary,proved that as a carrier for chemotherapeutic drugs,the stealth chitosan nanoparticles is more suitable.Methods:1.through combination of intermolecular hydrogen bonds of the negatively charged oxygen on PEG and positively amino charged on chitosan,and then prepare the PEG-modified drug-loaded nanoparticles by application of anion cohesion method. Use transmission electron microscopy to observation the morphological characteristics of the nanoparticles,laser particle size analyzer determined the particle size and distribution.UV Spectro-photometric for detect the drug loading efficiency. Dynamic dialysis study of drug-loaded nanoparticles in vitro drug release properties.2.Culture 5-8F nasopharyngeal carcinoma cells in vitro,and study different concentrations of drug-loaded nanoparticles,blank nanoparticles and free drug co-cultured with tumor cells after 4,48,72 hours on cell proliferation inhibition rate by MTT method.Calculated the drug concentration when the cell growth inhibition rate was 50%,which is IC50 values.3.Preparation of S-180 entities nude mice,using tail vein injection method of delivery,divided into stealth-based drug-loaded nanoparticles,the general drug-loaded nanoparticles,free drug and saline iv groups,the mice were killed one week after delivery,peeling the tumor,weighed,and calculated mass inhibition rate.4.Using SPSS 13.0 statistical software for data processing,all data expressed by mean±standard deviation(x±s),in vitro cell proliferation inhibition test using duplicate measurements of analysis;in vivo anti-tumor test using one-way ANOVA, homogeneity of variance by the Levene test,the use of Dunnett's method for multiple comparisons,P＜0.05 that the difference was significant.Results:1.The drug-loaded nanoparticles prepared were round or oval in shape,good dispersion when the CS/TPP mass ratio was 6:1,CS/EPI mass ratio was 8:1,the average particle size 322nm,drug loading was 13.9±1.1%,the encapsulation efficiency was 74.2±1.8%,72 hours cumulative release rate was 82%.2.Drug-loaded chitosan nanoparticles in vitro of 5-8F nasopharyngeal carcinoma cells growth inhibition with the concentration and time-dependent,with increasing drug concentration and extension of co-culture time,a gradual increase in inhibition rate,and the blank nanoparticles only at high concentrations when there is a certain degree of cell growth inhibition rate.3.Stealth chitosan nanoparticles group,ordinary chitosan nanoparticles and free drug group inhibition rate were significantly higher than saline control group(P＜0.05); compared with the free drug group,the chitosan nanoparticles encapsulated enhanced the anti-tumor effects of drugs(P＜0.01),compared with the general chitosan nanoparticles group,the stealth chitosan nanoparticles group has stronger anti-tumor effect(P=0.023).Conclusion:1.Chitosan nanoparticles loaded with epirubicin can be prepared by the anion cohesion method,and its have ideal sharp and particle size distribution,as well as high encapsulation efficiency and better drug controlled release properties.2.Stealth chitosan nanoparticles are expected to be a new type of chemotherapy drugs vector.
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